The core of many semi-synthetic cephalosporins, 7-aminocephalosporanic acid (7-ACA), can be manufactured chemically from cephalosporin C(CPC). The chemical process uses many chemical reagents that are highly toxic and heavily pollute the environment and is low in conversion rate and high in cost. Enzyme methods offer attractive alternative for production of fine chemicals without using toxic reagents and are high in conversion rate. The bioconversion of CPC to 7-ACA is conducted in two steps (FIG. 1): (1) CPC is first oxidized by D-amino acid oxidase to glutaryl-7-ACA; (2) glutaryl-7-ACA is in turn converted to 7-ACA by glutaryl-7-ACA acylase.
One of the major obstacles for large scale production of 7-ACA is the low yield and high cost of production of D-amino acid oxidase and glutaryl-7-ACA acylase. Current reports on the production level of D-amino acid oxidase is low, about 2,300 U/L fermentation medium (Pollegioni, L. et al., 1997, J. Biotechnol. 58, 115-123) and 800 U/L fermentation medium (Molla, G. et al., 1998, Protein Exp. Purif. 14, 289-294). The production level of glutaryl-7-ACA acylase production level is also low, about 129-2,500 U/L fermentation medium (Ishiye, M. and Niwa, M., 1992, Biochim. Biophys. Acta 1132, 233-239; Yang, Y. L. et al., 2001, CN1301813A; Xu, G. and Zhu, M. 2003, CN1428424A). Therefore, it is critical to produce these two enzymes for industrial production of 7-ACA at low cost.
Another obstacle for large scale production of 7-ACA resides in that existing manufacturing procedures are complex and expensive. For example, the procedures of related products by Roche Diagnostics (CC2 Twin Enzyme Process: D-AOD, product number: 1462865; Gl-Ac, product number: 1464213, Roche Diagnostics) are complicated (FIG. 1). Besides the reactions catalyzed by D-amino acid oxidase and glutaryl-7-ACA acylase, extra steps are needed: (1) due to the impurity of the D-amino acid oxidase used, a high proportion of α-ketoadipyl-7-ACA is not converted to glutaryl-7-ACA after the oxidation, thus an exogenous addition of hydrogen peroxide is required to complete the conversion; and (2) exogenous addition of catalase is required to degrade remaining hydrogen peroxide, for hydrogen peroxide can inactivate D-amino acid oxidase and oxidize CPC and glutaryl-7-ACA, thus reducing the final yield of 7-ACA.
In addition, a method to prepare 7-ACA has been published in CN1104255. In that method, since the expression vector used contained ampicillin resistance gene, the fermentation product contains β-lactamase, which significantly reduces the yield of 7-ACA and β-lactamase inhibitor is therefore required in the process. Furthermore, the host cell employed in that method produces catalase, which degrades hydrogen peroxide, therefore exogenous additions of hydrogen peroxide and catalase inhibitor are required. Consequently, the manufacturing procedures are complex and highly cost.